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The intra-S phase checkpoint and its effect on replication fork dynamics in saccharomyces cerevisiae

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THE INTRA-S PHASE CHECKPOINT AND ITS EFFECT ON REPLICATION FORK DYNAMICS IN SACCHAROMYCES CEREVISIAE
by
Shawn Joseph Szyjka
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
December 2008
Copyright 2008 Shawn Joseph Szyjka

Duplication of a cell's genetic material is of paramount importance. This task must be completed accurately, efficiently and occur once and only once within any given cell cycle. Origin "firing", which occurs according to a temporal program, results in the establishment of bidirectional replication forks. As replication forks traverse the chromosomal landscape, their stability is threatened by endogenous "obstacles" such as heavily transcribed tRNAs, protein-DNA complexes and "replication slow zones". In addition, fork stability can also be threatened by genotoxic agents. During S phase, in response to genotoxin-induced stress, the cell activates the intra-S phase checkpoint. The intra-S phase checkpoint is comprised of three major groups of proteins: sensors, adaptors and effectors. Sensor proteins detect problems at the replication fork and elicit a phosphorylation cascade that is mediated by adaptor proteins and results in the activation of effector kinases. Together, these proteins act to inhibit late origin firing, slow cell cycle progression, upregulate DNA repair genes, and stabilize replication forks until the stress has been alleviated.; In response to nucleotide depletion, the adaptor protein, Mrc1 (Mediator of the Replication Checkpoint), mediates a checkpoint signal that activates the effector kinase, Rad53. In addition to its checkpoint role, Mrc1 also appears to play a role in unperturbed DNA synthesis. Mrc1 travels with replication forks and mrc1delta cells display slowed bulk DNA synthesis. This phenotype could be due to decreased origin firing, increased fork pausing at endogenous "obstacles", or overall defective fork progression. Using two-dimensional (2D) gel electrophoresis of replication intermediates, we demonstrate that mrc1delta cells are not defective in fork pausing at tRNAs and that Mrc1's replication function is required for efficient progression of replication forks throughout the genome. We hypothesize that Mrc1 maintains association between polymerases and helicases at the replication fork, which results in efficient fork progression.; In response to MMS-induced DNA damage, Rad53 plays a vital role in replication fork stabilization. It is thought that Rad53 maintains the association of replisome components so that forks are poised for efficient restart upon checkpoint deactivation. Despite a wealth of knowledge with respect to checkpoint activation, relatively little is known about checkpoint deactivation and how replication forks restart. Previous work has suggested that replication fork progression along an MMS-damaged template is independent of the checkpoint. However, recent studies lacking Psy2-Pph3, a Rad53 phosphatase responsible for Rad53 deactivation after DNA damage, suggest otherwise, as the completion of S-phase after DNA damage is delayed in the absence of Psy2-Pph3. We have examined the role of Rad53 in the control of replication fork restart by monitoring BrdU incorporation at replication forks in the presence of DNA damage and during recovery from damage. We find that Rad53 deactivation is a key requirement for replication fork restart as cells lacking PPH3 are defective in fork progression in the presence of DNA damage and are delayed in replication restart during recovery. In addition, dominant-negative inactivation of Rad53 in pph3delta cells, enables replication fork restart, arguing that deactivation of Rad53 is sufficient for replication fork restart. Deletion of PTC2, which encodes a second, unrelated Rad53 phosphatase, in addition to PPH3, completely eliminates replication fork progression under DNA damaging conditions and results in lethality. These findings suggest that replication fork stabilization and restart involve a cycle of Rad53 activation and deactivation, and that at least two distinct phosphatases are responsible for regulating this process.

ii
THE INTRA-S PHASE CHECKPOINT AND ITS EFFECT ON REPLICATION FORK DYNAMICS IN SACCHAROMYCES CEREVISIAE
by
Shawn Joseph Szyjka
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
December 2008
Copyright 2008 Shawn Joseph Szyjka